Physical Chemistry
LD
Chemistry
Leaflets
Chemical equilibrium
Protolysis equilibrium
C4.2.2.1
Determination of the acidity constant of bromothymol blue
Aims of the experiment
 To get to know what an acid base indicator is and how it works.
Aims of the experiment
 To record and interpret absorbance spectra of dyes.
 To learn about a reaction with gases
 To investigate the pH dependency of the colour of bromothymol blue.
 To investigate the reduction of copper(II) oxide to copper or the oxidation of hydrogen to water
 To determine the acidity constant and the pKa value of bromothymol blue.
 To use and understand the Henderson-Hasselbalch equation.
Principles
When a chemical reaction comes to a halt, an equilibrium between educt and product is reached. Depending on the position
of a chemical equilibrium, small amounts of educts are still
present in the reaction mixture. Furthermore, where a chemical
equilibrium is concerned, we are dealing with a dynamic equilibrium. Although the proportion of product and educt in the
mixture is constant, the same amount of educt continues to
react to form product as product reacts back to form educt.
An acid base reaction is the prototype of a equilibrium reaction.
An equilibrium is generally reached very quickly with these
reactions. In a state of equilibrium, a substance is then present
in both a protonated and a non-protonated form. Depending on
the pH value, one form outweighs the other form. This can be
investigated using acid-base indicators.
If the relative concentrations of the protonated and the nonprotonated forms are known in the equilibrium, the pKa value
can be determined.
The indicator bromothymol blue used here is blue in alkaline
form and yellow in acid form. A green solution is obtained in the
neutral pH area due to there being a mixture of both forms. As
the absorption maxima of the two forms are far apart, the ratio
of concentration of both forms can be determined at every pH
value.
Absorbance spectra of bromothymol blue at various pH values
are therefore recorded in this experiment. For the evaluation,
the concentrations of both forms are determined using the
absorbance maxima, and from this the pKa value of bromothymol blue is calculated.
Acid-base indicators, e.g. bromothymol blue, are weak acids or
bases which have different colours depending on the pH value.
Different concentrations of both forms are present in a solution
depending on the pH value. The protonated form is denoted
HInd and the non-protonated form Ind . If acid is added to a
bromothymol blue solution, the concentration of the protonated
form HInd increases and that of the non-protonated form Ind
decreases. The colour of bromothymol blue changes from blue
(alkaline) to yellow (acidic). The equilibrium of the reaction
equation:
Ind
-
+
+H
blue
⇌
HInd
⇌
yellow
Fig. 1: Set-up of the experiment.
then lies on the left side.
As in all equilibrium reactions, the law of mass action can also
be employed:
SW-2014-06
KS 
[H ]  [Ind ]
[HInd]
Risk assessment
The substances used are in general non-hazardous. Also, it is
not necessary to wear gloves when working. It is recommended, however, to wear safety glasses.
Hydrochloric acid 0.1 mol/L
The acidity constant Ka is obtained in this way. However, in the
case of acids, the pKa value is usually stated, that is the negative decimal logarithm of the acidity constant. pKa values are
listed in tables and allow concentration-independent statements
to be made about the acid strength. The pH value is linked to
the pKa value via the Henderson-Hasselbalch equation.
[non - protonated form ]
[Ind  ]
pH  pK s  log
 pK s  log
[protonated form]
[HInd]
1
Hazard statements
H290 May be corrosive to metals.
Precautionary statements
P234 Keep only in original container.
Signal word:
Caution
P390 Absorb spillage to prevent material damage.
C4.2.2.1
LD Chemistry Leaflets
cylinder. First half-fill the measuring cylinder with water and
dissolve the substance using a glass rod. Then fill the measuring cylinder up to the 100 mL mark and pour the solution into a
labelled laboratory bottle.
Sodium hydroxide solution, 0.1 mol/L
Hazard statements:
H290 May be corrosive to metals.
Precautionary statements
Sodium dihydrogen phosphate solution (NaH2PO4, 0,1 mol/L):
For 100 mL of a 0.1 molar NaH2PO4 solution, weigh out 1.56 g
of sodium dihydrogen phosphate and place it in a measuring
cylinder. First half-fill the measuring cylinder with water and
dissolve the substance using a glass rod. Then fill the measuring cylinder up to the 100 mL mark and pour the solution into a
labelled laboratory bottle.
P234 Keep only in original container.
Signal word
Caution
P390 Absorb spillage to prevent material damage.
Sodium hydrogen phosphate
Hazard statements
Preparing the buffer solutions: From both phosphate solutions,
buffer solutions can now be made with various pH values, depending on how much of each of the two solutions is used. The
disodium hydrogen phosphate solution acts as a base and the
sodium dihydrogen phosphate solution as an acid. Buffers with
pH values 6.0, 7.0 and 8.0 are required for the experiment. For
this, transfer the volumes of the two solutions as shown in
Table 1 (measured using the measuring cylinders) into prepared and labelled laboratory bottles:
H318 Causes serious eye damage.
Precautionary statements
Signal word:
Hazard
P280 Wear
protective
gloves/protective clothing/eye protection/face protection.
P305+P351+P338 IF IN EYES: Rinse
continuously with water for several
minutes. Remove contact lenses if
present and easy to do. Continue rinsing.
Tab. 1: Preparation of the buffer solutions. Indication of the volumes of
both phosphate solutions needed for the appropriate pH values.
P313 Get medical advice/attention.
Bromothymol blue solution, 0.1 % in 20% ethanol
Contains ethanol at a non-hazardous
concentration
pH value
V Na2HPO4
V NaH2PO4
6.0
12 mL
88 mL
7.0
61 mL
39 mL
8.0
95 mL
5 mL
Now check the pH values with a pH meter. Calibrate this beforehand. If the pH value of the buffer solution is too great, add
the acid form of the buffer (NaH2PO4). If the pH value of the
buffer solution is too low, add the alkaline form (Na2HPO4).
Equipment and chemicals
1
2
Compact spectrometer USB, complete ......... 467 252
Rectangular cuvette, glass, 10 x 10 mm ....... 664 470
better: 5 pieces, or
5 Rectangular cuvette, plastic 10 x 10 mm ...... 6640474
1 Graduated pipette, 5 mL ............................... 665 996
1 Pipetting ball (Peleus ball) ............................ 666 003
5 Laboratory bottle, 100 mL, GL 45 ................ 602 345
2 Measuring cylinder, 100 mL .......................... 665 754
2 Glass rod, 200 mm x 5 mm diam. ................. 602 782
1 Electronic balance, 200 g: 0.01 g .................. 667 7977
1 Digital pH-meter 201 ..................................... 667 4781
1 Beaker, Boro 3.3, 100 mL, hF ....................... 664 137
1 Wash bottle, PE, 500 mL .............................. 661 243
1 Bromothymol blue solution, 50 mL ................ 671 0800
1 Hydrochloric acid 0.1 mol/L, 500 mL ............. 674 6950
1 Sodium hydroxide solution 0.1 mol/L, 500ml 673 8410
1 Disodium hydrogen phosphate, 250 g .......... 673 6710
1 Sodium dihydrogen phosphate, 250 g .......... 673 6010
1 Buffer solution set ......................................... 674 4600
1 Water, pure, 1L ............................................. 675 3400
Additionally required:
PC with Windows XP/Vista/7/8
Construction of the experiment
Prepare the solutions (phosphate buffer, hydrochloric acid,
sodium hydroxide solution and bromothymol blue solution), the
graduated pipette and the cuvettes.
Note: The experiment is easiest to perform if 5 glass cuvettes
are available. The alternatives are: 5 disposable cuvettes or 2
glass cuvettes which are rinsed with distilled water between the
individual measurements. It is not necessary to dry the cuvettes
as the solutions are buffered.
Connect the compact spectrometer to the computer with a USB
cable. Insert the cuvette holder.
Measuring with SpectraLab
1. Open SpectraLab.
2. Choose the "Intensity I1" display. Start the measurement
with
(in case this does not happen automatically).
Set-up and preparation of the experiment
Absorbance spectra of bromothymol blue at various pH values
are recorded in the experiment. For this, buffer solutions are
used having the pH values being investigated. These are mixed
in the cuvette with a bromothymol blue solution and absorbance spectra are recorded.
In preparation for this, buffer solutions with sodium phosphate
are initially prepared. However, other buffers with the appropriate pH values can also be used as an alternative.
Preparing the solutions
Disodium hydrogen phosphate solution. (Na2HPO4, 0.1 mol/L):
For 100 mL of a 0.1 molar Na2HPO4 solution, weigh out 1.42 g
of disodium hydrogen phosphate and place it in a measuring
2
3. Switch on the lamp
on the compact spectrometer. The
spectrum of the lamp is now visible. The maximum intensity
should be between 75 % and 100 %. This can be adjusted via
the integration time with
and
. The integration time is the
time during which light is collected which is then presented as
the measured value. The shorter the integration time, the lower
the intensity.
4. Place the black blocks (provided) into the cuvette holder to
record the background spectrum (offset). Open the "Offset I0"
display. The spectrum shown will be subtracted as the background spectrum during further measurements.
Note: SpectraLab always saves the last image produced in a
display. Therefore, to save the offset, for example, one only
needs to leave this display. Caution: If this is accessed again
during a later measurement, the previous offset value will be
replaced by the value being measured at the time.
C4.2.2.1
LD Chemistry Leaflets
5. Change back to the display "Intensity I1". An empty spectrum appears here.
6. Remove the black blocks. The spectrum of the lamp is now
visible again.
7. Fill a cuvette with water. This is the reference solution. Place
the cuvette into the compact spectrometer.
Note: Strictly speaking, the reference solution must also contain
the buffer substances. However, this is not necessary for
measurements of this accuracy.
8. Change to the display "Reference I2“. The spectrum shown
here serves as a reference spectrum for the following measurements.
9. Change to the display "Intensity I1". Here the spectrum obtained after the light passes through the solution can be seen.
The reference spectrum is also shown in grey.
10. Change to the display "Transmission T". This now shows
100 % at every wavelength. This is the maximum possible
amount of light . Any change in the amount of light is now
caused only by the dye in the solution.
11. Change to the display "Absorbance E". The absorbance
(optical density) is calculated and shown here. All further
measurements are made in this display.
Performing the experiment
Preparation of the solutions to be measured
1. Place three small drops of bromothymol blue solution into
each of the 5 cuvettes.
2. Into each of the cuvettes place 3 mL of the solutions of the
various pH values (hydrochloric acid, the buffer solutions with
pH values 6.0, 7.0 and 8.0, and sodium hydroxide solution).
Ensure that the solutions are mixed well.
Note: All cuvettes can be pipetted into using the same pipette.
Using water prepared ready in a beaker, rinse these once between the measurements. No changes in the pH value will
result owing to the effect of the buffer.
Recording the pH-dependent spectra of bromothymol blue.
Fig. 2: Spectra of bromothymol blue at pH 2 (yellow) and pH 10 (blue).
The solution with pH 6.0 already shows this second maximum
(see Fig. 3). The higher the pH value becomes, the more pronounced is this maximum. The solution with sodium hydroxide
solution (c. pH 10) alone produces a maximum at c. 615 nm
and has a minimum at c. 445 nm.
1. Insert the cuvettes one after the other into the compact
spectrometer. Start with the solution with the lowest pH value
(hydrochloric acid) and end with the solution with the highest
pH value (sodium hydroxide solution).
2. The spectrum of the solution appears as soon as the cuvette
is inserted. Save this by pressing the record button
. Then
insert the next cuvette.
Ending the measurements
1. End the measurements by pressing "Stop"
.
2. Switch off the lamp of the compact spectrometer by clicking
on
.
Fig. 3: Spectra of bromothymol blue at the pH values 2, 6, 7, 8 and 10.
A point at c. 497 nm can clearly be seen where all spectra
show the same absorbance.
Evaluation
Observation
The bromothymol blue solutions have different colours. The
hydrochloric acid solution is yellow. Colour sequences from
green to blue (sodium hydroxide solution) then follow.
Thus for the hydrochloric acid solution, a spectrum with a maximum at c. 430 nm is displayed in SpectraLab (see Fig. 2).
SpectraLab shows which wavelengths have been absorbed by
the coloured area under the curve. The yellow solution (hydrochloric acid) absorbs only blue wavelengths, the blue solution
(sodium hydroxide solution) absorbs mainly yellow and red
wavelengths. Here the maximum is at c. 615 nm.
3
The absorbance maxima
The maximum of the blue solution (in the yellow wavelength
range) is at c. 615 nm (see Fig. 3). The larger the amount of
blue in a solution, the greater is the maximum. If no blue is
contained in the solution, the absorbance at this wavelength will
be equal to 0.
The situation is different with the yellow solution. The absorbance maximum here is at λ = 440 nm. However, the absorbance minimum of the blue solutions is at λ = 445 nm. In order to
determine the ratio of the two forms of bromothymol blue in
each solution, this absorbance minimum is used.
C4.2.2.1
LD Chemistry Leaflets
-
The table with all spectra is copied into a spreadsheet program
for further evaluation. To do this, click with the right mouse
button on the table and choose "Copy table". Paste the data
into the spreadsheet.
Plot the absorbances of the spectra at λ = 445 nm and λ =
615 nm against the pH value (see Fig. 4). The absorbance at
445 nm corresponds to the concentration of the protonated
form HInd, which begins to fall from a pH value of 7. The absorbance at 615 nm corresponds to the non-protonated form
Ind, which then begins to rise analogously. At pH 2 the protonated form HInd is present 100 %, the non-protonated form Ind
is present 100 % at pH 10. At all other pH values, the ratio lies
between these extremes and can be calculated from the absorbance.
If the pH and the ratio of [Ind ]/[HInd] are known, the pKa value
can be determined from this equation. To do this, plot the pH
value against the logarithm of [Ind ]/[HInd]. A straight line is
produced whose intercept corresponds to the pKa value of
bromothymol blue (see Fig. 5)
615 nm
pH value
Calculating the pKa value of bromothymol blue
445
9
8
y = 0,5483x + 7,306
7
6
y = 0,8778x + 6,99
5
1,200
445 nm
1,000
-3
-2
615 nm
Absorbance
0,800
4
-1
0
log([Ind-]/[Hind]
1
2
Fig. 5: Plot of the pH value against log [Ind-]/[HInd]. Blue values:
measured at 615 nm, yellow values: measured at 445 nm.
0,600
The pKa value is 7.0 when measured at 615 nm and 7.3 when
measured at 445 nm.
0,400
Result
0,200
The colour of the solutions
0,000
1
2
3
4
5
6
7
8
9
10
11
pH value
Fig. 4: Plot of the absorbance against the pH value.
For the determination of the relative concentration, the absorbance at the end points (pH 2 and pH 10) is subtracted from all
other measured values. The absorbance is linear to the relative
concentration of both forms. The pKa value can therefore be
calculated using the values measured at 445 nm and 615 nm.
In order to calculate the ratio of Ind to HInd, the ratio is calculated between the absorbance at a certain pH value and the
maximum absorbance. At λ = 615 nm the following applies:
E  EpH2
[Ind ]

[HInd] EpH10  E
Where
E: is the absorbance (pH-dependent)
EpH2: is the absorbance at pH 2
EpH10: is the absorbance at pH 10
The calculation can be made analogously at λ = 445 nm:
EpH2  E
[Ind ]

[HInd] E  EpH10
The Henderson-Hasselbalch equation is:
pH  pK s  log
[Ind ]
[HInd]
The yellow solution (pH 2) absorbs at blue wavelengths (absorbance maximum c. 430 nm). The blue parts of the white light
are absorbed ("filtered out"). As a result, the solution appears in
the complementary colour yellow. This applies analogously to
the blue solution (pH 10). Here the yellow part of the light is
filtered out.
The isosbestic point
All spectra exhibit the identical absorbance at a certain wavelength. This point is referred to as the isosbestic point. The
absorbance at this point is always constant, independent of the
pH value. The isosbestic point is proof that in the case of these
various coloured solutions, we are dealing with just one dye.
This point is around λ = 497 nm for bromothymol blue.
The pKa value for bromothymol blue
By recording the spectra, it was possible to determine the pKa
value for the indicator bromothymol blue. It lies between 7.0
and 7.3. The literature value is 7.1 and so agrees with the values measured here.
The measurement becomes more precise if more values are
measured in the area of the point of inflection. To achieve this,
simply prepare more buffer solutions. The measurement itself
hardly takes any longer as a result.
Cleaning and disposal
The coloured solutions can be disposed of in the laboratory
drain with plenty of water. The prepared solutions can be used
for further experiments.
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